Affinity based molecular pull-down (immunoaffinity capture) and immunoprecipitation experiments are powerful and widely used methods to study the expression, modification and interaction of proteins in a wide variety of biological systems. Affinity based molecular pull-down and immunoprecipitation experiments have resulted in the rapid purification of epitope-tagged recombinant proteins, the discovery and characterization of reversible post-translational protein modifications, and a variety of other observations, which have increased the understanding of biological processes at the molecular level.
The underlying strategy behind the affinity based molecular pull-down technique is to use the affinity of biomolecules as a method to select target molecules from a solution. An affinity ligand, such as an antibody, is affixed to a particulate matrix, such as agarose, and the resulting conjugate is used to locate and bind its target, such as a particular antigen, from a biological or biochemical preparation.
Immunoprecipitation is a type of affinity based molecular pull down experiment. Affinity based molecular pull down experiments purify complexes using any affinity ligand that is directly conjugated to a polymeric support having a specific affinity for particular biomolecular targets. However, the general procedures of an affinity based molecular pull down experiment can be illustrated by the procedures involved in an immunoprecipitation experiment. The basic procedure of immunoprecipitation involves three stages. The first stage is preparation of the antigen solution. The second stage is preclearing the lysate of nonspecific background, and the third stage is forming and purifying the immune complexes. Once purified, any of a number of methods can be employed to analyze the antigens. The variety of procedures that can be used in affinity based molecular pull down and immunoprecipitation techniques specifically are described in detail in Harlow, E. and Lane, D. (eds.), “Antibodies: a Laboratory Manual”, Chapter 7, Cold Spring Harbor Press, NY (1988), the entirety of which is herein incorporated by reference.
Immunoprecipitation is usually performed on a lysate prepared from cells or tissue, although any aqueous solution can be used as a solution for performing an immunoprecipitation. Usually the lysate is prepared from the treatment of cells or tissue with some type of mild detergent. The mild detergent is effective in removing membranes, interfering with many weak intermolecular interactions and releasing most antigens from the cell, without disrupting the conformation or biochemical activity of the antigens of interest. Interferences from nonspecific binding proteins are minimized by pretreating the antigen solution with an antibody that does not bind the antigen of interest to remove the nonspecific binding proteins.
The immune complexes are formed by the addition of specific antibodies to the lysate. Antibodies have high affinity for their respective antigens so the antibody-antigen complexes form rapidly. The complexes are then purified by adding an affinity bead suspension, such as a protein A or protein G bead suspension, to the solution containing the antibody-antigen complexes. The purification occurs because protein A and protein G have a high affinity for the Fc portion of the antibody. After the complex is bound to the bead through the protein A/G-antibody interaction, the beads are collected by centrifugation and the unbound proteins are removed by washing the beads. The beads can be washed by rinsing with a solution such as lysis buffer and removing the lysate and wash buffer by aspiration. Complete removal of the wash buffer is important to lower the background and improve the effectiveness of the immunoassay. After completion of the formation and purification of the immune complexes, the resulting immunoprecipitated proteins can be further analyzed. Frequently, this next step is the separation of proteins by SDS-PAGE.
A major disadvantage of affinity based molecular pull down and immunoprecipitation procedures as commonly practiced is that the affinity matrix is difficult to visualize in the tubes used in the complex formation and purification steps. This difficulty in visualization leads to inefficient manipulations and results in loss of material and quantitative variability of results. For example, agarose or polyacrylamide beads generally are non-colored, i.e., translucent or white, and are difficult to visualize in the tubes, which makes procedural manipulations tedious. Due to this poor visibility, the beads can be lost during the aspiration of the lysate supernatant in the process of removal of the unbound proteins. Additionally, there can be accidental removal of the agarose or polyacrylamide beads from the tubes during the washing and aspiration steps due to the poor visibility of the beads. The difficulties and inaccuracies caused by the poor visibility of the beads are major limitations of the affinity based molecular pull down and immunoprecipitation experiments. Thus, a substantial need remains for an adaptation that would improve the visibility of the affinity matrix without altering its functioning in the molecular pull down or immunoprecipitation procedure. Such an adaptation would make the handling of the affinity matrix much easier for the individual conducting the molecular pull down or immunoprecipitation procedure. Improved handling would result in improved efficiency in manipulations and reduce the loss of material and quantitative variability of results. In short, such an adaptation would improve the efficiency and reliability of affinity based molecular pull down and immunoprecipitation procedures.
The affinity of certain dyes for proteins has been known and utilized for some time in various applications. Dyes have long been utilized as affinity ligands for the purification of proteins in affinity chromatography. Affinity chromatography is a procedure which separates a substance from a mixture by virtue of the biospecific recognition and affinity (involving noncovalent interactions) of that substance for a ligand immobilized to a support. The dyes that have been employed to purify proteins were originally developed for use in the textile industry. These dyes include triazine dyes that are based on the chemistry of cyanuric chloride (1,3,5-trichlorotriazine). Triazine dyes have been used as ligands for affinity chromatography because they offer advantages in both preparation and use over more conventional immobilized coenzyme and various other biological group-specific media. These advantages include a protein binding capacity that can be significantly higher than that of natural biospecific media, low cost, general availability and ease of preparation. See, for example, Christopher R. Lowe and James C. Pearson, Affinity Chromatography on Immobilized Dyes, Methods In Enzymology, Vol. 104, 97-113 (1984).
U.S. Pat. No. 5,597,485 discloses a process for separating proteins using a polymeric composition that includes a polymer formed from at least one monomer containing a polymerizable moiety chemically bonded to a synthetic anionic organic dye. The dye has an affinity for the protein to be separated. The process for separating proteins includes retaining the protein on the dye fraction and then recovering the protein from the polymer. The process is applicable to affinity chromatography but is not limited to chromatographic columns. The process includes contacting the polymer with the solution containing the protein to be separated and separating the colored particles from the solution by means of filtration, centrifugation, or similar means. The process, however, deliberately utilizes dyes possessing a specific affinity for the protein to be separated and the protein is isolated and purified by binding specifically to this dye.
U.S. Pat. No. 4,546,161 discloses a process of producing affinity chromatography media by reacting mono or di-chloro triazine dyes with a solid support matrix possessing free hydroxy or amino groups. The solid support matrix is a polymer or copolymer of agarose, dextrose, dextran or acrylamide. The dyes are reacted in the presence of an alkali metal hydroxide and an alkali metal salt. The dyes are linked to the solid support matrix via the triazine ring, providing high and specific protein-binding capacity. The media are designed specifically for use in affinity chromatography for the efficient and highly specific isolation and purification of proteins using triazine dyes selected to specifically bind the proteins of interest.
Similarly, U.S. Pat. No. 4,880,915 discloses a process for purifying a specific protein, human TNF, comprising applying a solution containing human TNF to a column packed with a dye-bonded crosslinked agarose gel, eluting the column to release the bound human TNF, and recovering a column fraction of purified human TNF. The crosslinked agarose gel comprises a crosslinked agarose gel support to which a dye ligand is covalently bonded. The dye ligand is either Cibacron Blue F3GA, Procion Red HE3B or Green A and the human TNF is retained by specifically interacting with the dye ligand.
Dyes have also been employed for their ability to enhance the visibility of a substance in the specific application of nucleic acid precipitation. WO 97/12994 discloses a method for precipitating soluble nucleic acid from a solution by adding a polymeric carrier molecule that is coupled to an indicator molecule. The technique of nucleic acid precipitation is often prone to unpredictable failure due to loss of precipitated nucleic acid pellets during the removal of supernatant phases, due to the lack of visibility of the nucleic acid pellets. The nucleic acids are precipitated by adding a sufficient amount of salt and alcohol to the aqueous solution to cause the nucleic acids to precipitate out of solution. Alcohol and salt are common ingredients for the technique of nucleic acid precipitation, and ethanol is the alcohol most commonly used in the procedure.
The modified carrier molecule disclosed in WO 97/12994 has the same solubility and precipitation properties as the unmodified molecule and co-precipitates with the nucleic acid, but is readily visualized and enables the user to observe the location of nucleic acid in the treated sample. The solubility and precipitation properties of both the unmodified and modified molecule are specific to the precipitation and centrifugation techniques generally employed in nucleic acid precipitation. Specifically, nucleic acids are soluble in more aqueous solutions and aggregate when the dielectric constant of the aqueous solutions is lowered, such as by adding alcohol to the aqueous solution. The aggregation causes the nucleic acids to collectively possess sufficient mass to precipitate out of the aqueous solution. The precipitate has to be separated from the aqueous solution by subjecting the solution to high speed centrifugation of around 5000 g for a period of time of around five minutes. Thus, the modified carrier molecule would not be employable in alternative experiments, and there is no suggestion in WO 97/12994 that the modified carrier molecule would be applicable to alternative techniques. Rather, the method and carrier molecule are limited specifically to nucleic acid precipitation techniques.